The pyridine nucleotide NAD is directly involved in the regulation of cell growth. We observe that carcinogenic N-nitroso compounds cause a rapid depression of cellular NAD. This depression appears to be related to NAD-dependent nuclear modification reactions associated with carcinogen-induced damage to DNA and/or repair of DNA damage. A major objective of this proposal is to study in detail the ability of N-nitroso compounds of known carcinogenicity to cause acute depression of NAD in 3T3 cells and in mitogen stimulated human lymphocytes. Several classes of direct-acting and indirect-acting carcinogens will be examined to determine whether acute depression of NAD is a general effect of carcinogens. The extent of DNA damage caused by N-nitroso compounds and the rate of DNA repair of carcinogen induced DNA damage will be studied in normal and NAD depleted 3T3 cells. Sensitive, quantitative methods involving gas chromatography-mass spectrometry and high pressure liquid chromatography for measuring NAD-dependent nuclear modifications as mono and poly adenosine diphosphoribose (ADPR) will be developed. The extent of these NAD-dependent nuclear modifications will be studied in normal cells and in cells exposed to carcinogenic N-nitroso compounds. Carcinogen induced DNA damage and the rate of DNA repair will be studied in the presence of the methylated xanthines caffine and theophylline which are specific inhibitors of NAD-dependent modifications.